KR102402276B1 - Fusion protein comprising modified interleukin-7 and tgf-beta receptor ii and use thereof - Google Patents

Abstract

The present invention relates to a fusion protein comprising a modified interleukin-7 and TGF beta receptor II and uses thereof, and the fusion protein has a high production yield and can effectively inhibit cancer, so it is useful for the treatment of cancer or infectious diseases can be used

Description

Fusion protein comprising modified interleukin-7 and TGF beta receptor II and use thereof

The present invention relates to a fusion protein comprising a modified interleukin-7 and TGF beta receptor II and uses thereof.

TGF beta receptor II (transforming growth factor beta receptor II, TBRII) is a protein encoded by the TGFBR2 gene in humans. It is a membrane protein of 70 to 80 kDa. TBRII forms a heterodimer with TBRI (TGF beta receptor I) and regulates transcription of genes related to cell proliferation by binding to TGF-α and transmitting intracellular signals. TBRII is composed of a C-terminal protein kinase domain and an N-terminal ecto domain. The ecto domain is a domain of a membrane protein that extends into the extracellular space and forms a folded structure comprising a single helix stabilized by 9 beta chains and 6 disulfide bonds in the strand.

In addition, TGF-α, known as a ligand of TBRII, is an immunosuppressive cytokine overexpressed in cancer cells. It is known as one of the mechanisms of immune evasion of cancer cells, such as inhibition of T-cell proliferation due to TGF-α secreted from cancer cells.

Recently, research on the decoy receptor of TGF-α including a fragment of TBRII is being actively conducted, but there is a problem in that the intracellular activity and stability of the fragment of TBRII are poor.

On the other hand, interleukin-7 (interleukin 7, IL-7) is a cytokine that promotes an immune response mediated by B cells and T cells, and particularly plays an important role in the adaptive immune system. Specifically, IL-7 activates the immune function by promoting the survival and differentiation of T cells and B cells, the survival of lymphoid cells, and the activation of natural killer cells (NK cells). It is important for the development of cells and B cells. It binds to hepatocyte growth factor (HGF) and is a pre-pro-B cell growth-stimulating factor, a cofactor of the V(D)J rearrangement of T cell receptor beta (TCRβ). ) (Muegge K, 1993, Science 261 (5117): 93-5).

However, when producing recombinant IL-7 for pharmaceutical use, there are problems in that a large amount of impurities are produced compared to a general recombinant protein, denaturation is easy, and mass production is not easy.

Muegge K, 1993, Science 261 (5117): 93-5

An object of the present invention is to provide a fusion protein comprising a modified interleukin-7 (IL-7) and TGF beta receptor II (TBRII).

Another object of the present invention is to provide an isolated nucleic acid molecule encoding the fusion protein.

Another object of the present invention is to provide an expression vector comprising the nucleic acid molecule.

Another object of the present invention is to provide a host cell comprising the expression vector.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer or infectious disease comprising the fusion protein as an active ingredient.

In order to achieve the above object, the present invention provides a fusion protein comprising a modified interleukin-7 (IL-7) and TGF beta receptor II (TBRII).

The invention also provides an isolated nucleic acid molecule encoding the fusion protein.

In addition, the present invention provides an expression vector comprising the nucleic acid molecule.

In addition, the present invention provides a host cell comprising the expression vector.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer or infectious disease comprising the fusion protein as an active ingredient.

The fusion protein comprising the modified interleukin-7 (IL-7) and TGF beta receptor II (TBRII) of the present invention has a high production yield and can effectively inhibit cancer, which can be usefully used in the treatment of cancer or infectious diseases. can

1 shows the gene constructs of sTBRII-hyFc-IL7 and IL7-hyFc-sTBRII.
2 is a result of measuring the production of sTBRII-hyFc-IL7 and IL7-hyFc-sTBRII fusion proteins through batch culture.
Figure 3 is the result of measuring the in vivo activity of the sTBRII-hyFc-IL7 fusion protein according to the dose, (a) is after administration of the sTBRII-hyFc-IL7 fusion protein to a mouse animal model, the number of CD8+ T cells The measurement result, (b) is the result of measuring the CD8+ T cell increase rate on the 7th day of administration, (c) is the result of measuring the number of CD4+ T cells, (d) is the CD4+CD25+ Foxp3+ Treg cell is the result of measuring the number of , (e) is the result of measuring the number of neutrophils, (f) is the result of measuring the number of NK cells (○: PBS; ■: sTBRII-hyFc-IL7, 10 mpk; ▲: sTBRII-hyFc-IL7, 30 mpk; ▼: sTBRII-hyFc-IL7, 100 mpk).
Figure 4 is the result of measuring the in vivo activity of the sTBRII-hyFc-IL7 fusion protein according to the administration route, (a) is a mouse animal model after administration of the sTBRII-hyFc-IL7 fusion protein, immune cells (lymphocytes) It is the result of measuring the number, and (b) is the result of measuring the increase rate of immune cells different from intravenous and subcutaneous administration on the 7th day of administration.
Figure 5 is the result of analyzing the in vivo activity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins, (a) is after administration of the sTBRII-hyFc-IL7 fusion protein to a mouse tumor model, CD8+ T The result of measuring the number of cells, (b) is the result of measuring the increase rate of CD8+ T cells on the 7th day of administration, (c) is the result of administering the IL-7-hyFc-sTBRII fusion protein to the mouse tumor model, The result of measuring the number of CD8+ T cells, (d) is the result of measuring the increase rate of CD8+ T cells on the 7th day of administration.
6 is a result of measuring the concentration of TGF beta in the serum after administration of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins to a mouse tumor model.
7 is a result of measuring tumor volume after administration of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins to a mouse tumor model.
8 is a result showing (a) cell proliferation and (b) its standard curve after treatment with sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in 2E8 cell line.
9 is a result showing the standard curve of the luminescence intensity of the SBE reporter after the SMAD Signaling Pathway SBE Reporter-HEK293 cell line was treated with sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins.
10 is a result of comparative analysis of the simultaneous binding ability of TGF-β1 and IL-7Rα of each fusion protein, according to the concentration of IL-7Rα (2nd ligand) after binding to TGF-β1 (1st ligand) (a) sTBRII-hyFc-IL7 and (b) IL-7-hyFc-sTBRII fusion protein binding affinities were measured.
11 is a result of comparative analysis of the simultaneous binding capacity of TGF-β1 and IL-7Rα of each fusion protein, according to the concentration of TGF-β1 (2nd ligand) after binding to IL-7Rα (1st ligand) (a) sTBRII-hyFc-IL7 and (b) IL-7-hyFc-sTBRII fusion protein binding affinities were measured.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a fusion protein comprising a modified interleukin-7 (interleukin 7, IL-7) and TGF beta receptor II (transforming growth factor beta receptor II, TBRII).

The modified IL-7 may have the following structure:

A - IL-7;

In this case, A is an oligopeptide composed of 1 to 10 amino acid residues, and the modified IL-7 is IL-7 or a polypeptide having a similar activity.

As used herein, the term "IL-7 or a polypeptide having a similar activity" refers to a polypeptide or protein having the same or similar sequence and activity to IL-7.

The IL-7 may include an IL-7 protein or a fragment thereof. In this case, IL-7 may be derived from humans, rats, mice, monkeys, cattle, or sheep.

Specifically, human IL-7 may have the amino acid sequence of SEQ ID NO: 1 (Genbank Accession No. P13232); Rat IL-7 is Genbank Accession No. may have the amino acid sequence disclosed in P56478; Mouse IL-7 is Genbank Accession No. It may have an amino acid sequence disclosed at P10168; Monkey IL-7 is Genbank Accession No. may have the amino acid sequence disclosed in NP_001279008; Bovine IL-7 is Genbank Accession No. have the amino acid sequence disclosed in P26895; Both IL-7 were identified as Genbank Accession No. It may have the amino acid sequence disclosed in Q28540.

The IL-7 may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. In addition, the modified IL-7 is about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 from the sequence of SEQ ID NO: 1 %, 98% or 99% or more.

In addition, the IL-7 protein or fragment thereof may include variously modified proteins or peptides, ie, variants. The modification may be performed through a method of substituting, deleting or adding one or more proteins to wild-type IL-7 without altering the function of IL-7. These various proteins or peptides can be combined with wild-type proteins by 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % or more.

Usually, substitution of wild-type amino acid residues is alanine, but can be carried out by conservative amino acid substitutions that do not affect or weaken the charge of the whole protein, ie, polarity or hydrophobicity.

Reference may be made to Table 1 below for conservative amino acid substitutions.

basic Arginine (Arg, R)
Lysine (Lys, K)
Histidine (His, H) acid Glutamic acid (Glu, E)
Aspartic acid (Asp, D) uncharged polar Glutamine (Gln, O)
Asparagine (Asn, N)
Serine (Ser, S)
Threonine (Thr, T)
Tyrosine (Tyr, Y) non-polar Phenylalanine (Phe, F)
Tryptophan (Trp, W)
Cysteine (Cys, C)
Glycine (Gly, G)
Alanine (Ala, A)
Val, V
Proline (Pro, P)
Methionine (Met, M)
Leu (Leu, L)
norleucine
isoleucine

For each amino acid, additional conservative substitutions include "homologs" of the amino acid. In this case, the "homolog" refers to an amino acid in which a methylene group (CH ₂ ) is inserted into the side chain at the beta position of the side chain of the amino acid. Examples of such "homologs" include, but are not limited to, homophenylalanine, homoarginine, homoserine, and the like. As used herein, the term "IL-7 protein" is also used as a concept including "IL-7 protein and fragments thereof". The terms “protein,” “polypeptide,” and “peptide” may be used interchangeably unless otherwise specified.

In the structure of the modified IL-7, A may be directly linked to the N-terminus of IL-7, or linked through a linker. The A may be linked to the N-terminus of IL-7. The A is characterized in that it contains 1 to 10 amino acids, and the amino acids may be selected from the group consisting of methionine, glycine, and combinations thereof.

Methionine and glycine do not induce an immune response in the body. Protein therapeutics produced from E. coli necessarily contain methionine at the N-terminus, but no adverse immune side effects have been reported. In addition, glycine is widely used as a GS linker, but even commercially available products such as Dulaglutide do not induce an immune response ( Cell Biophys. 1993 Jan-Jun:22(1-3):189- 224).

According to one embodiment, A may be an oligopeptide including 1 to 10 amino acids selected from the group consisting of methionine (Met, M), glycine (Gly, G), and combinations thereof. Preferably, it may be an oligopeptide containing 2 to 10 amino acids, and more preferably, an oligopeptide containing 3 to 10 amino acids, but is not limited thereto. Specifically, A is methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, Methionine-glycine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, glycine-glycine-glycine, methionine-methionine-methionine-methionine, methionine-glycine-methionine-methionine, methionine-glycine-glycine-methionine, methionine- Glycine-Glycine-Glycine, Methionine-Glycine-Methionine-Glycine, Glycine-Methionine-Methionine-Methionine, Glycine-Methionine-Glycine-Glycine, Glycine-Glycine-Glycine-Glycine, Methionine-Methionine-Methionine-Methionine-Methionine, Methionine- Methionine-glycine-methionine-methionine, methionine-methionine-glycine-glycine-methionine, methionine-glycine-methionine-methionine-glycine, methionine-methionine-methionine-methionine-glycine, glycine-glycine-glycine-glycine-glycine, glycine- Glycine-methionine-methionine-methionine, glycine-glycine-glycine-methionine-glycine, methionine-glycine-methionine-glycine-methionine-glycine, methionine-methionine-methionine-glycine-glycine-glycine, methionine-methionine-glycine-glycine- Methionine-methionine, glycine-glycine-methionine-methionine-glycine-glycine, methionine-glycine-methionine-glycine-methionine-glycine-methionine-glycine, methionine-methionine-methionine-methionine-glycine-glycine-glycine-glycine, methionine- Methionine-glycine-glycine-methionine-methionine-glycine-glycine, methionine-methionine-methionine-methionine-glycine-glycine-glycine-glycine, methionine-glycine-methionine-glycine-methionine-glycine-methionine-glycine-methionine-glycine or It may have one N-terminal sequence selected from the group consisting of methionine-methionine-methionine-methionine-methionine-glycine-glycine-glycine-glycine-glycine.

The modified IL-7 may consist of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

As used herein, the term "TBRII" or "TGF beta receptor II" refers to any vertebrate, including mammals, e.g., primates (e.g. humans) and rodents (e.g. mice and rats), unless otherwise stated. Any wild-type TBRII obtained from an animal source. The human TBRII may consist of the amino acid sequence of SEQ ID NO: 4. Specifically, the TBRII may be an extracellular domain of TBRII. The extracellular domain of TBRII may have the amino acid sequence of positions 24 to 159 of human TBRII (SEQ ID NO: 4), and the extracellular domain of TBRII may consist of the amino acid sequence of SEQ ID NO: 5.

As used herein, the term “sTBRII” refers to soluble TBRII, and may be an extracellular domain of human TBRII.

The modified IL-7 and TBRII may be bound by an immunoglobulin Fc domain.

The Fc domain may be wild-type or mutant. The Fc domain variant may be the Fc domain of a modified immunoglobulin. In this case, the Fc domain of the modified immunoglobulin has a modified binding ability with an Fc receptor and/or complement, such that antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) This may be weakened. The modified immunoglobulin may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and combinations thereof. In particular, the Fc domain of the modified immunoglobulin may include a hinge region, a CH2 domain and a CH3 domain in an N-terminal to C-terminal direction. In this case, the hinge region comprises a human IgD hinge region, the CH2 domain comprises a portion of an amino acid residue of a CH2 domain of human IgD and a portion of an amino acid residue of a CH2 domain of a human IgG4, wherein the CH3 domain is a portion of an amino acid residue of a human IgG4 and a portion of the amino acid residues of the CH3 domain. The hinge region may be an IgG1 hinge region, which may include the amino acid sequence of SEQ ID NO: 6.

As used herein, the term "Fc domain", "Fc fragment" or "Fc" includes the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin, but includes the variable regions and light chains of its heavy and light chains. It refers to a protein that does not contain the constant region 1 (CL1). It may further comprise a hinge region of the heavy chain constant region. Hybrid Fc or hybrid Fc fragments are also referred to herein as “hFc” or “hyFc”.

As used herein, the term "Fc domain variant" refers to one in which some amino acids in the Fc domain are substituted or prepared by combining different types of Fc domains. The Fc domain variant can prevent cleavage at the hinge region. Specifically, the 144th amino acid and/or the 145th amino acid of SEQ ID NO: 9 may be modified. Preferably, the 144th amino acid K of SEQ ID NO: 9 may be substituted with G or S (K144G, K144S), and the 145th amino acid E may be substituted with G or S (E145G, E145S).

In addition, the Fc domain or Fc domain variant of the modified immunoglobulin may be represented by the following formula (I):

[Formula (I)]

N'-(Z1)p-Y-Z2-Z3-Z4-C'

In the above formula,

N' is the N-terminus of the polypeptide and C' is the C-terminus of the polypeptide;

p is an integer equal to 0 or 1; and

Z1 is an amino acid sequence having 5 to 9 consecutive amino acid residues in the N-terminal direction from position 98 among the amino acid residues at positions 90 to 98 of SEQ ID NO: 7,

Y is an amino acid sequence having 5 to 64 consecutive amino acid residues in the N-terminal direction from position 162 among the amino acid residues at positions 99 to 162 of SEQ ID NO: 7,

Z2 is an amino acid sequence having 4 to 37 consecutive amino acid residues in the C-terminal direction from position 163 among the amino acid residues at positions 163 to 199 of SEQ ID NO: 7,

Z3 is an amino acid sequence having 71 to 106 consecutive amino acid residues in the N-terminal direction from position 220 among the amino acid residues at positions 115 to 220 of SEQ ID NO: 8;

Z4 is an amino acid sequence having 80 to 107 amino acids in the C-terminal direction from position 221 among the amino acid residues at positions 221 to 327 of SEQ ID NO: 8.

In addition, the Fc fragment of the present invention may be in the form of a native sugar chain, an increased sugar chain compared to the native type, a decreased sugar chain compared to the native type, or a form in which the sugar chain is removed. Immunoglobulin Fc sugar chains can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Removal of sugar chains from the Fc fragment sharply reduces the binding affinity of the primary complement component C1 to C1q and results in a decrease or loss of ADCC or CDC, thereby not inducing an unnecessary immune response in vivo. In this regard, the immunoglobulin Fc fragment in a deglycosylated or aglycosylated form may be more suitable for the purpose of the present invention as a drug carrier. As used herein, the term “deglycosylation” refers to enzymatic removal of sugars from an Fc fragment. In addition, the term "aglycosylation" means that the Fc fragment is produced in an unglycosylated form by prokaryotes, preferably E. coli .

In addition, the Fc domain of the modified immunoglobulin comprises the amino acid sequence of SEQ ID NO: 9 (hyFc), SEQ ID NO: 10 (hyFcM1), SEQ ID NO: 11 (hyFcM2), SEQ ID NO: 12 (hyFcM3) or SEQ ID NO: 13 (hyFcM4) can do.

According to the present invention, the Fc domain of the modified immunoglobulin may be that described in U.S. Patent No. 7,867,491, and the production of the Fc domain of the modified immunoglobulin may be performed with reference to that described in U.S. Patent No. 7,867,491. .

In addition, the fusion protein may be one in which TBRII, an Fc domain, and modified IL-7 are combined in order from the N-terminus to the C-terminus. Such a fusion protein may be denoted as "sTBRII-hyFc-IL7".

In the case of the sTBRII-hyFc-IL7 fusion protein, a first linker may be further included between the TBRII and Fc domains. The first linker may consist of 20 to 60 consecutive amino acids, or 25 to 50 consecutive amino acids, or 30 to 40 amino acids. In one embodiment, the first linker may consist of 20 amino acids. In addition, the first linker may include (G4S)n (in this case, n is an integer of 1 to 5). In one embodiment of the present invention, the first linker may consist of the amino acid sequence of SEQ ID NO: 14.

In addition, a second linker may be further included between the Fc domain and the modified IL-7. The second linker may consist of 1 to 30 consecutive amino acids, or 3 to 20 consecutive amino acids, or 4 to 16 amino acids. In addition, the second linker may include (SG3)n (in this case, n is an integer of 1 to 5). In one embodiment of the present invention, the second linker may consist of the amino acid sequence of SEQ ID NO: 15.

Accordingly, the sTBRII-hyFc-IL7 fusion protein may have the following structure.

N' - TBRII - (L1)p - Fc domain - (L2)q - A-IL-7 - C'

above,

N' is N-terminal, C' is C-terminal,

L1 is a first linker, L2 is a second linker,

p and q are integers equal to 0 or 1.

The sTBRII-hyFc-IL7 fusion protein may consist of the amino acid sequence of SEQ ID NO: 17. The fusion protein is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Or it may have a sequence having at least 99% homology.

In addition, the fusion protein may be one in which the modified IL-7, Fc domain, or TBRII is sequentially bound from the N-terminus to the C-terminus. Such a fusion protein may be denoted as "IL7-hyFc-sTBRII".

In the case of the IL7-hyFc-sTBRII fusion protein, a first linker may be further included between the modified IL-7 and the Fc domain. The first linker may consist of 20 to 60 consecutive amino acids, or 25 to 50 consecutive amino acids, or 30 to 40 amino acids. In one embodiment, the first linker may consist of 20 amino acids. In addition, the first linker may include (G4S)n (in this case, n is an integer of 1 to 5). In one embodiment of the present invention, the first linker may consist of the amino acid sequence of SEQ ID NO: 14.

In addition, a third linker may be further included between the Fc domain and TBRII. The third linker may consist of 1 to 30 consecutive amino acids, or 3 to 20 consecutive amino acids, or 4 to 16 amino acids. In one embodiment of the present invention, the third linker may consist of the amino acid sequence of SEQ ID NO: 16.

Accordingly, the IL7-hyFc-sTBRII fusion protein may have the following structure.

N' - A-IL-7 - (L1)p - Fc domain - (L3)r - TBRII - C'

above,

N' is N-terminal, C' is C-terminal,

L1 is a first linker, L2 is a second linker,

p and r are integers equal to 0 or 1.

The IL7-hyFc-sTBRII fusion protein may consist of the amino acid sequence of SEQ ID NO: 18. The fusion protein is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NO: 18, Or it may have a sequence having at least 99% homology.

The present invention also provides a nucleic acid molecule encoding the fusion protein.

In addition, the nucleic acid molecule may additionally include a signal sequence (or signal peptide) or a leader sequence.

As used herein, the term "signal sequence (or signal peptide)" refers to a short peptide present at the N-terminus of a newly synthesized protein classified into a secretory pathway. Signal sequences useful in the present invention include an antibody light chain signal sequence, such as antibody 1418 (Gillies et al., J Immunol Meth 1989 125: 191-202), an antibody heavy chain signal sequence, such as the MOPC141 antibody heavy chain signal sequence (Sakano). et al, Nature 1980 286: 676-683), and other signal sequences known in the art (see, eg, Watson et al, Nucleic Acid Research 1984 12:5145-5164).

The signal peptide is well known in the art for its characteristics, and is generally known to include 16 to 30 amino acid residues, and may include more or fewer amino acid residues. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region.

The central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence through the membrane lipid bilayer during migration of the immature polypeptide. After initiation, the signal sequence is cleaved in the lumen of the ER by cellular enzymes commonly known as signal peptidases. In this case, the signal sequence may be a secretion signal sequence of tPa (tissue Plasminogen Activation), HSV gDs, or growth hormone. Preferably, the secretion signal sequence used in higher eukaryotic cells, including mammals, may be used, and more preferably, the tPa sequence (SEQ ID NO: 19) or the amino acid sequence of SEQ ID NO: 20 may be used. In addition, the signal sequence of the present invention can be used by substituting a codon having a high expression frequency in the host cell.

In addition, the present invention provides an expression vector comprising the nucleic acid molecule.

As used herein, the term "vector" is understood as a nucleic acid means comprising a nucleotide sequence that can be introduced into a host cell, recombination and insertion into the host cell genome, or can replicate spontaneously as an episome. The vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

As used herein, the term "host cell" refers to prokaryotic and eukaryotic cells into which recombinant expression vectors can be introduced.

In the present invention, an appropriate host cell can be transformed or transfected with the DNA sequence of the present invention, and can be used for expression and/or secretion of a target protein. Presently preferred host cells that can be used in the present invention are immortal hybridoma cells, NS/0 myeloma cells, 293 cells, Chinese hamster ovary cells (CHO cells), HeLa cells. , CapT cells (human amniotic fluid derived cells) and COS cells.

As used herein, the terms “transformation” and “transfection” refer to the introduction of a nucleic acid (eg, a vector) into a cell by a number of techniques known in the art.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer or infectious disease comprising a fusion protein comprising a modified IL-7 and TBRII as an active ingredient.

The cancer is gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma consisting of It may be selected from the group, but is not limited thereto.

The infectious disease may be one selected from the group consisting of hepatitis B, hepatitis C, human papilloma virus infection, cytomegalovirus infection, viral respiratory disease, and influenza, but is not limited thereto.

In the composition for treating or preventing cancer or infectious disease of the present invention, the active ingredient may be used in any amount (effective amount) according to the use, formulation, purpose of formulation, etc., as long as it exhibits anticancer activity or a therapeutic effect on infectious diseases. may be included, but a typical effective amount will be determined within the range of 0.001% to 20.0% by weight based on the total weight of the composition. Here, "effective amount" refers to an amount of an active ingredient capable of inducing an anticancer effect or an infectious disease treatment effect. Such an effective amount can be determined empirically within the ordinary ability of one of ordinary skill in the art.

In this case, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any non-toxic material suitable for delivery to a patient. Distilled water, alcohol, fats, waxes and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

Specifically, the pharmaceutical composition may be prepared as a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond what the application (prescription) target can adapt.

When the pharmaceutical composition is prepared for parenteral use, it may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories together with suitable carriers according to methods known in the art. When formulating as an injection, a suitable carrier may be sterile water, ethanol, polyol such as glycerol or propylene glycol, or a mixture thereof, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine or sterile water for injection. , an isotonic solution such as 5% dextrose may be used. Formulation of pharmaceutical compositions is known in the art, and specifically, reference may be made to the literature [Remington's Pharmaceutical Sciences (19th ed., 1995)] and the like. This document is considered a part of this specification.

A preferred dosage of the pharmaceutical composition is in the range of 0.01 ug/kg to 10 g/kg, or 0.01 mg/kg to 1 g/kg per day, depending on the patient's condition, weight, sex, age, patient's severity, and administration route. can be Administration may be performed once or divided into several times a day. Such dosages should not be construed as limiting the scope of the invention in any respect.

Subjects to which the pharmaceutical composition can be applied (prescribed) are mammals and humans, particularly preferably humans. In addition to the active ingredient, the pharmaceutical composition of the present application may further include any compound or natural extract known to have an anticancer activity or therapeutic effect on an infectious disease, and safety has already been verified for the increase/reinforcement of anticancer activity.

In addition, the present invention provides the use of a fusion protein comprising the modified IL-7 and TBRII for producing a pharmaceutical preparation having a preventive or therapeutic effect on cancer or infectious disease.

In addition, the present invention provides a method for preventing or treating cancer or an infectious disease comprising a fusion protein comprising modified IL-7 and TBRII as an active ingredient.

The therapeutically effective amount includes the specific composition including the type and extent of the response to be achieved, whether or not other agents are used if necessary, the individual's age, weight, general health, sex and diet, administration time, administration route, and composition. It is preferable to apply differently depending on various factors including the secretion rate, treatment period, and drugs used or concurrently with a specific composition and similar factors well known in the pharmaceutical field. Therefore, it is preferable to determine the effective amount of the composition suitable for the purpose of the present invention in consideration of the foregoing.

The subject is applicable to any mammal, and the mammal includes not only humans and primates, but also domestic animals such as cattle, pigs, sheep, horses, dogs and cats.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of sTBRII-hyFc-IL7 and IL7-hyFc-sTBRII fusion proteins

The present inventors fused IL-7 or sTBRII to the N-terminus of the Fc domain to prepare a fusion protein in which human-derived IL-7 and sTBRII (soluble TGF beta receptor II) are bound, and to the C-terminus of the Fc domain A gene construct fused with IL-7 or sTBRII was prepared. In the present invention, the fusion protein in which TBRII, Fc domain and IL-7 are coupled in order from N-terminus to C-terminal direction is denoted as "sTBRII-hyFc-IL7", and IL-7, Fc domain or TBRII is N- The fusion protein bound in order from the end to the C-terminus was denoted as "IL7-hyFc-sTBRII".

First, in the preparation of the sTBRII-hyFc-IL7 gene construct, sTBRII uses only the extracellular domain of 24-159 aa in the known amino acid sequence of TGF beta receptor II (Accession number: NP003233.4). was fused to the N-terminus of the Fc domain, and IL-7 was fused to the C-terminus of the Fc domain using a known amino acid sequence (Accession number: NP000871.1) to prepare a gene construct (Fig. 1). ).

In addition, in the preparation of the IL7-hyFc-sTBRII gene construct, IL-7 and sTBRII were fused to the N-terminus and C-terminus of the Fc domain using the same sequence as above, respectively, to form the gene construct. prepared (Fig. 1).

IL-7, sTBRII and Fc domains were prepared as sub-vectors by synthesizing each gene in Cosmogene Tech, and Golden GATEway assembly was performed to prepare three synthesized gene segments into one gene segment. In the process, an expression vector was obtained as a pGP30 vector.

The expression vector was transfected with the Neon Transfection system (Invitrogen, MPK1096) into CHO cells (suspension-adapted Chinese Hamster Ovary cells) adapted to suspension culture, and highly productive cell lines were obtained through HT selection and Methotrexate (Sigma, M8407) amplification. was selected. In addition, a single cell was obtained through limiting dilution cloning to obtain a production cell line producing sTBRII-hyFc-IL7 and IL7-hyFc-sTBRII fusion proteins.

Each production cell line was cultured in 80 mL increments (batch culture) using a 250 mL Erlenmeyer flask (Corning, 431144) using Hycell CHO medium (Hyclone, SH30949.02) to confirm the culture productivity. As a result, it was confirmed that the IL7-hyFc-sTBRII fusion protein was produced in an amount of 0.6 g/L, and the sTBRII-hyFc-IL7 fusion protein was produced in an amount of 0.95 g/L ( FIG. 2 ).

Example 2. In vivo (in vivo) of sTBRII-hyFc-IL7 fusion protein according to the dose in vivo ) activity assay

The present inventors performed an experiment to confirm the pharmacodynamic profile (pharmacodynamics profile) of the fusion protein according to the intravenous administration. Briefly, after intravenous administration of sTBRII-hyFc-IL7 fusion protein at 10, 30 and 100 mg/kg (mpk) to a C57BL/6 (B6) mouse animal model, changes in immune cells in the blood over time were analyzed. In addition, blood was collected through retro-orbital bleeding on days 3, 7, 10, 14, 17 and 21 after administration of the sTBRII-hyFc-IL7 fusion protein. Each value was calculated by checking the expression rate of the cell surface indicator and the composition ratio of each cell in the blood through flow cytometry, and multiplying it with a value measured using CBC (complete blood count).

As a result, in mice administered with the sTBRII-hyFc-IL7 fusion protein, the number of CD8 ⁺ T cells, the main target cells of IL-7, increased in a concentration-dependent manner, reaching a peak on the 7th day of administration ( Fig. 3a). In addition, it was confirmed that the increase rate of CD8 ⁺ T cells on the 7th day of administration increased by 6.22, 12.45 and 35.04 times at concentrations of 10, 30 and 100 mpk, respectively, compared to the control group (PBS treatment) ( FIG. 3b ).

In addition, in mice administered with the sTBRII-hyFc-IL7 fusion protein, the IL-7 target cells, CD4 ⁺ T cells and CD4 + CD25 + Foxp3 + Treg cells, also showed a tendency to increase ( FIGS. 3c and 3d ). However, no change according to administration was observed in neutrophils and NK cells other than the target cells ( FIGS. 3e and 3f ).

Example 3. In vivo (in vivo) of sTBRII-hyFc-IL7 fusion protein according to administration route in vivo ) activity assay

The present inventors performed an experiment to measure the in vivo activity of the sTBRII-hyFc-IL7 fusion protein according to the administration route. Briefly, sTBRII-hyFc-IL7 fusion protein (10 mpk) was administered to a C57BL/6 (B6) mouse animal model intravenously (intravenous injection, i.v.) or subcutaneously (subcutaneous injection, s.c.), followed by time-dependent blood immunity Cell changes were analyzed. Blood was collected through retro-orbital bleeding on days 0, 4, 7, 11, and 14 after administration of the sTBRII-hyFc-IL7 fusion protein. Each level was measured using a complete blood count (CBC).

As a result, it was confirmed that the number of immune cells (lymphocytes) increased in the mice administered with the sTBRII-hyFc-IL7 fusion protein compared to the control group (PBS-treated) on the 7th day of administration (FIG. 4a). Specifically, on the 7th day of administration, it was confirmed that the number of immune cells increased by about 1.51 times compared to the control group in the intravenous administration group, and the number of immune cells increased by about 3.04 times compared to the control group in the subcutaneous administration group (FIG. 4b).

Example 4. In vivo (in vivo) of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a mouse tumor model in vivo ) activity assay

The present inventors performed an experiment to analyze the in vivo activity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a mouse tumor model. Briefly, a mouse tumor model was prepared by subcutaneously injecting 1x10 ⁵ MC38 colorectal cancer cell lines into a C57BL/6 (B6) mouse animal model. Then, after subcutaneous administration (sc) of each fusion protein at 10 mpk or 20 mpk on the 6th day after tumor formation, changes in immune cells in the blood were analyzed using flow cytometry and complete blood count (CBC). did Blood was collected by retro-orbital bleeding on days 0, 3, 7, 11, 15, and 18 after administration of each fusion protein.

As a result, in tumor mice administered with the sTBRII-hyFc-IL7 fusion protein, the cell number of CD8 ⁺ T cells increased in a concentration-dependent manner, and reached a peak point on the 7th day of administration ( FIG. 5a ). In addition, it was confirmed that the CD8 ⁺ T cell increase rate on the 7th day of administration was increased to 11.08 and 21.82 times at concentrations of 10 and 20 mpk, respectively, compared to the control group (PBS treatment) ( FIG. 5b ).

In addition, in tumor mice administered with the IL-7-hyFc-sTBRII fusion protein, the number of CD8 ⁺ T cells increased in a concentration-dependent manner, and reached a peak point on the 7th day of administration ( FIG. 5c ). In addition, it was confirmed that the CD8 ⁺ T cell increase rate on the 7th day of administration was increased by 18.74 and 29.56 times at concentrations of 10 and 20 mpk, respectively, compared to the control group (PBS treatment) ( FIG. 5D ).

From the above results, it was confirmed that both the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins had the activity of increasing the proliferation of target cells such as CD8 ⁺ T cells in a concentration-dependent manner.

Example 5. TGF beta inhibitory activity analysis of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a mouse tumor model

The present inventors performed an experiment to analyze the inhibitory activity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins on TGF beta in a mouse tumor model. Briefly, a mouse tumor model was prepared by subcutaneously injecting 1x10 ⁵ MC38 colorectal cancer cell lines into a C57BL/6 (B6) mouse animal model. Then, on the 6th day after the tumor was formed, each fusion protein was subcutaneously administered (sc) at 20 mpk. Blood was collected through retro-orbital bleeding at 2, 6, 24, 48, 72 and 168 hours after administration of each fusion protein, and serum was separated from the collected blood samples. The concentration of TGF beta present in the serum was measured using the Mouse TGF-beta 1 DuoSet ELISA Kit (R&D systems, catalog# DY1679-05).

As a result, in tumor mice administered with the sTBRII-hyFc-IL7 fusion protein, TGF beta in the serum was inhibited for about 48 hours after administration, detected again 72 hours after administration, and 168 hours after administration, control group (PBS treatment) It was confirmed that it was recovered to a level similar to that of (FIG. 6). In addition, in tumor mice administered with the IL7-hyFc-sTBRII fusion protein, TGF beta in the serum was inhibited for about 72 hours after administration, and it was confirmed that 168 hours after administration, it was recovered to a level similar to that of the control group (PBS treatment) (Fig. 6).

From the above results, it was confirmed that the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins had the effect of inhibiting TGF beta in the serum for 48 to 72 hours through subcutaneous administration.

Example 6. Analysis of anticancer activity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a mouse tumor model

The present inventors performed an experiment to confirm the anticancer activity of the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a mouse tumor model. Briefly, a mouse tumor model was prepared by subcutaneously injecting 1x10 ⁵ MC38 colorectal cancer cell lines into a C57BL/6 (B6) mouse animal model. Then, on the 6th day after the tumor was formed, each fusion protein was subcutaneously administered (sc) at 20 mpk. Changes in tumor volume were measured at intervals of 2-3 days after administration. The tumor volume was calculated using the following formula.

Tumor volume = {(Long axis length) x (Short axis length) ² } / 2

In addition, the tumor growth inhibition rate (% tumor growth inhibition) was calculated using the following formula (formula).

% tumor growth inhibition (% TGI) = {(MTVcontrol - MTVtreated) / MTVcontrol} x 100 (* MTV = median tumor volume)

As a result, it was confirmed that the mice administered with the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins had the effect of significantly reducing the tumor volume compared to the control group (PBS treatment) (FIG. 7). In particular, on the 20th day after transplantation of the MC38 colorectal cancer cell line, in mice administered with the sTBRII-hyFc-IL7 fusion protein, the tumor growth inhibition rate was 61.79% compared to the control group (Fig. 7a), and IL7-hyFc-sTBRII fusion In mice administered with the protein, the tumor growth inhibition rate was 83.14% compared to the control group (FIG. 7b).

From the above results, it was confirmed that the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins had significant anticancer efficacy through subcutaneous administration.

Example 7. In vitro ( in vitro ) Analysis of IL-7 bioactivity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins

In order to measure the biological activity of IL-7 constituting the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in vitro, the present inventors reported that the IL-7-dependently proliferating 2E8 cell line (ATCC ^® TIB -239 ^TM ( Mouse B lymphocyte cell line) was used for the experiment. Since human-derived IL-7 has cross-species reactivity with mice, it is possible to induce proliferation of the mouse cell line 2E8 cell line by human-derived IL-7.

Briefly, after thawing the frozen 2E8 cell line, stabilized cells were prepared by subculture at least 3 times in a T-75 flask. The 2E8 cell line was cultured using IMDM (ATCC ^® 30-2005 ^TM ) medium containing mouse IL-7 (Cell Signaling, 5217SC) and FBS (Hyclone, SH30084.03). Thereafter, the cultured 2E8 cell line was suspended in IMDM medium not containing mouse IL-7 to make it starvation for IL-7, and then aliquoted at 1x10 ⁵ cells/well in a 96-well plate. Thereafter, cells were treated with sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a concentration gradient sequentially diluted from 3 nM to 1/3, respectively, in a 37°C, 5% CO ₂ incubator for 3 days. cultured.

Cell proliferation was quantified using CellTiter 96® AQ _ueous One Solution Assay (Promega, G3581). Cells were treated with MTS reagent, 37° C., 5% CO ₂ After incubation for 4 hours in an incubator, absorbance at 490 nm wavelength was measured using an ELISA plate reader. Using the GraphPad Prism ^® program (GraphPad Software), the standard curve of absorbance and the EC ₅₀ (50 % effective concentration) value of the two fusion proteins were calculated based on this.

As a result, the EC ₅₀ of the sTBRII-hyFc-IL7 fusion protein was measured to be 52.52 pM, and the EC ₅₀ of the IL-7-hyFc-sTBRII fusion protein was measured to be 56.21 pM ( FIG. 8 and Table 2).

EC ₅₀ (pM) sTBRII-hyFc-IL-7 IL-7-hyFc-sTBRII 52.52 56.21

Example 8. in vitro ( in vitro ) Analysis of TGF beta inhibitory activity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in

In order to measure the biological activity of sTBRII constituting the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins, the present inventors developed SMAD Signaling, which can measure the sub-transmission signal induced by TGF beta as a luminescent signal. Experiments were performed using the Pathway SBE Reporter-HEK293 cell line (BPS Bioscience, 60653).

Briefly, the SMAD Signaling Pathway SBE Reporter-HEK293 cell line was cultured in a 37° C., 5% CO ₂ incubator using Growth Medium 1B (BPS Bioscience, 79531) containing Geneticin (Invitrogen, 11811031). Thereafter, the cultured SMAD Signaling Pathway SBE Reporter-HEK293 cell line was suspended in Assay Medium 1B (BPS Bioscience, 79617-2) and aliquoted at 3.5x10 ⁴ cells/well in a white clear-bottom 96-well microplate. Thereafter, cells were treated with sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a concentration gradient sequentially diluted from 50 nM to 1/3, respectively, and at the same time 20 ng/mL of TGF beta (BPS Bioscience, 90900-1) after treating the cells together, 37 ℃, 5% CO ₂ Incubated in an incubator for 18 hours. After the incubation, the cells were treated with ONE-Step ^TM Luciferase reagent and incubated by shaking the plate at room temperature for 30 minutes. Then, the luminescence degree of the SBE reporter was measured with a plate-reading luminometer (TECAN SPARK 10M). Using the GraphPad Prism ^® program (GraphPad Software), the standard curve of the luminescence of the SBE reporter and the IC ₅₀ values of the two fusion proteins were calculated.

As a result, in the SMAD Signaling Pathway SBE Reporter-HEK293 cell line, the IC _{50 value of the sTBRII-hyFc-IL-7 fusion protein was measured to be 1.874 nM, and the IC 50 value} of the IL-7-hyFc-sTBRII fusion protein was measured to be 0.4148 nM. (Fig. 9 and Table 3).

IC ₅₀ (nM) sTBRII-hyFc-IL-7 IL-7-hyFc-sTBRII 1.874 0.4148

Example 9. Simultaneous binding analysis of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins

The present inventors performed BLI (biolayer interferometry) to compare and analyze the simultaneous binding capacity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII to IL-7Rα (CD127) and TGF-β1. Experiments were performed using an amine reactive second-generation biosensor (AR2G) and an AR2G reagent kit (AR2G Reagent Kit).

Each fusion protein was mixed with recombinant human TGF-β1 protein (2 μg/mL) (immobilized ligand (1 ^st ligand)) - fusion protein (1000 nM) - recombinant human IL-7R α protein (1,2, 4, 8 μg) /mL) (2 ^nd ligand) in that order; or recombinant human IL-7R α protein (2 μg/mL) (immobilized ligand (1 ^st ligand))-fusion protein (1000 nM)-recombinant human TGF-β1 protein (0.25, 0.5, 1, 2 μg/mL) ( 2 ^nd ligand), the samples were added in that order, and the simultaneous binding and binding according to the concentration of the 2 ^nd ligand of the fusion protein bound to the 1 ^st ligand were compared and analyzed.

As a result of confirming the binding of each fusion protein to the immobilized ^1st ligand (TGF-β1, IL-7R α ), the association was good over time, and the binding rates of both fusion proteins were similar. In addition, it was confirmed that relatively binding to TGF-β1 was better than binding to IL-7R α .

After binding to TGF-β1 (1 ^st ligand), binding to the fusion protein according to the concentration of IL-7R α (2 ^nd ligand) was confirmed. As a result, it was confirmed that the concentration-dependent increase in both fusion proteins was confirmed. (Fig. 10). In addition, after binding to IL-7R α (1 ^st ligand), binding to the fusion protein according to the concentration of TGF-β1 (2 ^nd ligand) was confirmed. As a result, both fusion proteins increased in a concentration-dependent manner (Fig. 11).

From the above results, it was confirmed that the sTBRII-hyFc-IL-7 and IL-7-hyFc-sTBRII fusion proteins were capable of simultaneous binding of the target proteins TGF-β1 and IL- 7Rα .

<110> Genexine, Inc. POSTECH ACADEMY-INDUSTRY FOUNDATION <120> FUSION PROTEIN COMPRISING MODIFIED INTERLEUKIN-7 AND TGF-BETA RECEPTOR II AND USE THEREOF <130> 1068276 <150> KR 10-2019-0146805 <151> 2019-11-15 <160> 20 < 170> KoPatentIn 3.0 <210> 1 <211> 177 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for human IL-7 <400> 1 Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile 1 5 10 15 Leu Val Leu Leu Pro Val Ala Ser Ser Asp Cys Asp Ile Glu Gly Lys 20 25 30 Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile Asp Gln Leu 35 40 45 Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe 50 55 60 Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu Gly Met Phe 65 70 75 80 Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys Met Asn Ser 85 90 95 Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu Gly Thr Thr 100 105 110 Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys Pro Ala Ala 115 120 125 L eu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu 130 135 140 Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu 145 150 155 160 Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly Thr Lys Glu 165 170 175 His <210> 2 <211> 155 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for modified IL-7(MGM-IL-7) <400> 2 Met Gly Met Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu 1 5 10 15 Ser Val Leu Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu 20 25 30 Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His 35 40 45 Ile Cys Asp Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg 50 55 60 Lys Leu Arg Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu 65 70 75 80 His Leu Leu Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr 85 90 95 Gly Gln Val Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro 100 105 110 Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu 115 120 125 Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys 130 135 140 Trp Asn Lys Ile Leu Met Gly Thr Lys Glu His 145 150 155 <210> 3 <211> 180 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for modified IL-7(signal peptide-MGM-IL-7) <400> 3 Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile 1 5 10 15 Leu Val Leu Leu Pro Val Ala Ser Ser Met Gly Met Asp Cys Asp Ile 20 25 30 Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile 35 40 45 Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn 50 55 60 Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu 65 70 75 80 Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys 85 90 95 Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu 100 105 110 Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys 115 120 125 Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn 130 135 140 Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys 145 150 155 160 Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly 165 170 175 Thr Lys Glu His 180 <210> 4 <211> 567 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for TGF beta receptor II <400> 4 Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val 20 25 30 Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 35 40 45 Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 50 55 60 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 65 70 75 80 Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 85 90 95 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 100 105 110 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 115 120 125 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 130 135 140 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu 145 150 155 160 Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu 165 170 175 Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn 180 185 190 Arg Gln Gln Lys Leu Ser Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys 195 200 205 Leu Met Glu Phe Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg 210 215 220 Ser Asp Ile Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu 225 230 235 240 Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gly Lys Gly Arg Phe Ala 245 250 255 Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu 260 265 270 Thr Val Ala Val Lys Ile Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys 275 280 285 Thr Glu Lys Asp Ile Phe Ser Asp Ile Asn Leu Lys His Glu Asn Ile 290 295 300 Leu Gln Phe Leu Thr Ala Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln 305 310 315 320 Tyr Trp Leu Ile Thr Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr 325 330 335 Leu Thr Arg His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser 340 345 350 Ser Leu Ala Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro Cys 355 360 365 Gly Arg Pro Lys Met Pro Ile Val His Arg Asp Leu Lys Ser Ser Asn 370 375 380 Ile Leu Val Lys Asn Asp Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu 385 390 395 400 Ser Leu Arg Leu Asp Pro Thr Leu Ser Val Asp Asp Leu Ala Asn Ser 405 410 415 Gly Gln Val Gly Thr Ala Arg Tyr Met Ala Pro Glu Val Leu Glu Ser 420 425 430 Arg Met Asn Leu Glu Asn Val Glu Ser Phe Lys Gln Thr Asp Val Tyr 435 440 445 Ser Met Ala Leu Val Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val 450 455 460 Gly Glu Val Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu 465 470 475 480 His Pro Cys Val Glu Ser Met Lys Asp Asn Val Leu Arg Asp Arg Gly 485 490 495 Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn His Gln Gly Ile Gln Met 500 505 510 Val Cys Glu Thr Leu Thr Glu Cys Trp Asp His Asp Pro Glu Ala Arg 515 520 525 Leu Thr Ala Gln Cys Val Ala Glu Arg Phe Ser Glu Leu Glu His Leu 530 535 540 Asp Arg Leu Ser Gly Arg Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp 545 550 555 560 Gly Ser Leu Asn Thr Thr Lys 565 <210> 5 <211> 137 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for sTBRII (extracellular domain) <400> 5 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp Leu 130 135 <210> 6 <211> 16 <212> PRT < 213> Artificial Sequence <220> <223> amino acid sequence for IgG1 hinge <400> 6 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 7 <211> 384 <212 > PRT <213> Artificial Sequence <220> <223> amino acid sequence for human IgD constant region (Genbank accession No. P01880) <400> 7 Ala Pro Thr Lys Ala Pro Asp Val Phe Pro Ile Ile Ser Gly Cys Arg 1 5 10 15 His Pro Lys Asp Asn Ser Pro Val Val Leu Ala Cys Leu Ile Thr Gly 20 25 30 Tyr His Pro Thr Ser Val Thr Val Thr Trp Tyr Met Gly Thr Gln Ser 35 40 45 Gln Pro Gln Arg Thr Phe Pro Glu Ile Gln Arg Arg Asp Ser Tyr Tyr 50 55 60 Met Thr Ser Ser Gln Leu Ser Thr Pro Leu Gln Gln Trp Arg Gln Gly 65 70 75 80 Glu Tyr Lys Cys Val Val Gln His Thr Ala Ser Lys Ser Lys Lys Glu 85 90 95 Ile Phe Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro 100 105 110 Thr Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala 115 120 125 Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys 130 135 140 Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu 145 150 155 160 Cys Pro Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala 165 170 175 Val Gln Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val 180 185 190 Val Gly Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly 195 200 205 Lys Val Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser 210 215 220 Asn Gly Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu 225 230 235 240 Trp Asn Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu 245 250 255 Pro Pro Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro 260 265 270 Val Lys Leu Ser Leu Asn Leu Leu Ala Ser Asp Pro Glu Ala 275 280 285 Ala Ser Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile 290 295 300 Leu Leu Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe 305 310 315 320 Ala Pro Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala 325 330 335 Trp Ser Val Leu Arg Val Pro Ala Pro Ser Pro Gln Pro Ala Thr 340 345 350 Tyr Thr Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala 355 360 365 Ser Arg Ser Leu Glu Val Ser Tyr Val Thr Asp His Gly Pro Met Lys 370 375 380 <210> 8 <211> 327 <212> PRT <213> Artificial Sequence <220> < 223> amino acid sequence for Partial human IgG4 constant region (Genbank accession No. AAH25985) <400> 8 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Val Leu Asp Ser Asp Gly Ser Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 9 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for hyFc <400> 9 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys 1 5 10 15 Glu Glu Gin Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys 245 <210> 10 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for hyFcM1 <400> 10 Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 15 Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 20 25 30 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 35 40 45 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 50 55 60 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 65 70 75 80 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 85 90 95 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 115 120 125 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 130 135 140 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 145 150 155 160 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 165 170 175 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 180 185 190 Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 195 200 205 Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 11 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for hyFcM2 <400> 11 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys 245 <210> 12 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for hyFcM3 <400> 12 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Ser Gly Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Se r 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys 245 <210> 13 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for hyFcM4 <400> 13 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Ser Ser Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys 245 <210> 14 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for linker 1 <400> 14 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 15 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for linker 2 <400> 15 Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 <210> 16 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for linker 3 <400> 16 Gly Gly Gly Gly Ser Gly 1 5 <210> 17 <211> 582 <212> PRT <213> Artificial Sequence <220> <223> amino acid for sTBRII-hyFc-IL7 <400> 17 Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser His Ala Ile Pro Pro His Val Gln Lys 20 25 30 Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys 35 40 45 Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp 50 55 60 Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu 65 70 75 80 Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn 85 90 95 Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp 100 105 110 Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys 115 120 125 Lys Lys Pro Gly Glu Thr Phe Phe Phe Met Cys Ser Cys Ser Ser Asp Glu 130 135 140 Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro 145 150 155 160 Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 165 170 175 Gly Gly Gly Gly Ser Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 180 185 190 Cys Pro Pro Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe 195 200 205 Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val 210 215 220 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 225 230 235 240 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 245 250 255 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 260 265 270 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 275 280 285 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 290 295 300 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 305 310 315 320 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 325 330 335 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 340 345 350 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 355 360 365 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 370 375 380 Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His 385 390 395 400 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Ser Gly Gly Gly Ser 405 410 415 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Met Gly Met Asp Cys 420 425 430 Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val 435 440 445 Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys 450 455 460 Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn 465 470 475 480 Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe 485 490 495 Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val 500 505 510 Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly 515 520 525 Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu 530 535 540 Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe 545 550 555 560 Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu 565 570 575 Met Gly Thr Lys Glu His 580 210> 18 <211> 567 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for IL-7-hyFc-sTBRII <400> 18 Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile 1 5 10 15 Leu Val Leu Leu Pro Val Ala Ser Ser Met Gly Met Asp Cys Asp Ile 20 25 30 Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile 35 40 45 Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn 50 55 60 Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu 65 70 75 80 Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys 85 90 95 Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu 100 105 110 Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys 115 120 125 Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn 130 135 140 Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys 145 150 155 160 Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly 165 170 175 Thr Lys Glu His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 180 185 190 Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 195 200 205 Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys 210 215 220 Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 225 230 235 240 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 245 250 255 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 260 265 270 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 275 280 285 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 290 295 300 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 305 310 315 320 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 325 330 335 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 340 345 350 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 355 360 365 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 370 375 380 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 385 390 395 400 Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln 405 410 415 Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly Ser Gly Ile 420 425 430 Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp 435 440 445 Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val 450 455 460 Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser 465 470 475 480 Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp 485 490 495 Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro 500 505 510 Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys 515 520 525 Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys 530 535 540 Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu 545 550 555 560 Tyr Asn Thr Ser Asn Pro Asp 565 <210> 19 <211> 25 <212> PRT <213> Artificial Sequence <220> < 223> amino acid sequence for human IL-7 <400> 19 Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser His Ala 20 25 <210> 20 < 211> 25 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for signal peptide of IL-7 <400> 20 Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile 1 5 10 15Leu Val Leu Leu Pro Val Ala Ser Ser 20 25

Claims (27)

Interleukin-7 (interleukin 7, IL-7) and TGF beta receptor II (transforming growth factor beta receptor II, TBRII), including, SEQ ID NO: 17 or fusion protein consisting of the amino acid sequence of SEQ ID NO: 18.
delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete An isolated nucleic acid molecule encoding the fusion protein according to claim 1 .
delete An expression vector comprising the nucleic acid molecule according to claim 21 .
A host cell comprising the expression vector of claim 23 .
A pharmaceutical composition for preventing or treating colon cancer comprising the fusion protein according to claim 1 as an active ingredient.
delete delete

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